Endoderm, cardiac and neural inducing factors - human frazzled (frzb-1) protein

ABSTRACT

Novel proteins have been designated “cerberus” and “frzb-1,” respectively. Cerebus is expressed as a secreted peptide during embryogenesis of the Xenopus embryo, and is expressed specifically in the head organizer region. This new molecule has endodermal, cardiac, and neural tissue inducing activity, that should prove useful in therapeutic, diagnostic, and clinical applications requiring regeneration, differentiation, or repair of these and other tissues. Frzb-1 is a soluble antagonist of growth factors of the Wnt family that acts by binding to Wnt growth factors in the extracellular space. A third novel protein is therm PAPC which promotes the formation of dorsal mesoderm and somites in the embryo.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/020,150, filed Jun. 20, 1996.

[0002] This invention was made with Government support under grantcontract number HD-21502, awarded by the National Institutes of Health.The Government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] The invention generally relates to growth factors, neurotrophicfactors, and their inhibitors, and more particularly to several newgrowth factors with neural, endodermal, and cardiac tissue inducingactivity, to complexes and compositions including the factors, and toDNA or RNA coding sequences for the factors. Further, one of the novelgrowth factors should be useful in tumor suppression gene therapy.

BACKGROUND OF THE INVENTION

[0004] Growth factors are substances, such as polypeptide hormones,which affect the growth of defined populations of animal cells in vivoor in vitro, but which are not nutrient substances. Proteins involved inthe growth and differentiation of tissues may promote or inhibit growth,and promote or inhibit differentiation, and thus the general term“growth factor” includes cytokines, trophic factors, and theirinhibitors.

[0005] Widespread neuronal cell death accompanies normal development ofthe central and peripheral nervous systems. Studies of peripheral targettissues during development have shown that neuronal cell death resultsfrom the competition among neurons for limiting amounts of survivorfactors (“neurotrophic factors”). The earliest identified of these,nerve growth factor (“NGF”), is the most fully characterized and hasbeen shown to be essential for the survival of sympathetic and neuralcrest-derived sensory neurons during early development of both chick andrat.

[0006] One family of neurotropic factors are the Wnts, which have dorsalaxis-inducing activity. Most of the Wnt proteins are bound to cellsurfaces. (See, e.g., Sokol et al., Science, 249, pp. 561-564, 1990.)Dorsal axis-inducing activity in xenopus embryos by one member of thisfamily (Xwnt-8) was described by Smith and Harland in 1991, Cell, 67,pp. 753-765. The authors described using RNA injections as a strategyfor identifying endogenous RNAs involved in dorsal patterning to rescuedorsal development in embryos that were ventralized by UV irradiation.

[0007] Another member of the growth and neurotropic factor family wassubsequently discovered and described by Harland and Smith, which theytermed “noggin.” (Cell, 70, pp. 829-840 (1992).) Noggin is a goodcandidate to function as a signaling molecule in Nieuwkoop's center, byvirtue of its maternal transcripts, and in Spemann's organizer, throughits zygotic organizer-specific expression. Besides noggin, othersecreted factors may be involved in the organizer phenomenon.

[0008] Another Xenopus gene designated “chordin ” that begins to beexpressed in Spemann's organizer and that can completely rescue axialdevelopment in ventralized embryos was described by Sasai et al., Cell,79, pp. 779-790, 1994. In addition to dorsalizing mesoderm, chordin hasthe ability to induce neural tissue and its activities are antagonizedby Bone Morphogenetic Protein-4 (Sasai et al., Nature, 376, pp. 333-336,1995).

[0009] Therefore, the dorsal lip or Spemann's organizer of the Xenopusembryo is an ideal tissue for seeking novel growth and neurotrophicfactors. New growth and neurotrophic factors are useful agents,particularly those that are secreted due to their ability to be used inphysiologically active, soluble forms because these factors, theirreceptors, and DNA or RNA coding sequences therefore and fragmentsthereof are useful in a number of therapeutic, clinical, research,diagnostic, and drug design applications.

SUMMARY OF THE INVENTION

[0010] In one aspect of the present invention, the sequence of the novelpeptide that can be in substantially purified form is shown by SEQ IDNO: 1. The Xenopus derived SEQ ID NO: 1 has been designated “cerberus,”and this peptide is capable of inducing endodermal, cardiac, and neuraltissue development in vertebrates when expressed. The nucleotidesequence which, when expressed results in cerberus, is illustrated bySEQ ID NO: 2. Since peptides of the invention induce endodermal,cardiac, and neural tissue differentiation in vertebrates, they shouldbe able to be prepared in physiologically active form for a number oftherapeutic, clinical, and diagnostic applications.

[0011] Cerberus was isolated during a search for molecules expressedspecifically in Spemann's organizer containing a secretory signalsequence. In addition to cerberus, two other novel cDNAs wereidentified.

[0012] The Xenopus derived peptide that can be deduced from SEQ ID NO: 3encodes a novel protein we had earlier designated as “frazzled,” asecreted protein of 318 amino acids that has dorsalizing activity inXenopus embryos. We now designate the novel protein as “frzb-1.” Thegene for frzb-1 is expressed in many adult tissues of many animals,three of the cDNAs (Xenopus, mouse, and human) have been cloned by us.The accession numbers for the Xenopus, mouse, and human frzb-1 cDNAsequences of the gene now designated frzb-1 are U68059, U68058, andU68057, respectively. Frzb-1 has some degree of sequence similarity tothe Drosophila gene frizzled which has been shown to encode aseven-transmembrane protein that can act both as a signalling and as areceptor protein (Vinson et al., Nature, 338, pp. 263-264, 1989; Vinsonand Adler, Nature, 329, pp. 549-551, 1987). Vertebrate homologues ofFrizzled have been isolated and they too were found to be anchored tothe cell membrane by seven membrane spanning domains (Wang et al., J.Biol. Chem., 271, pp. 4468-4476, 1996). Frzb-1 differs from the frizzledproteins in that it is an entirely soluble, diffusible secreted proteinand therefore suitable as a therapeutic agent. The nucleotide sequencederived from Xenopus that, when expressed, results in frzb-1 protein isillustrated by SEQ ID NO: 4. The frzb-1 protein derived from mouse isshown as SEQ ID NO: 7, while the mouse frzb-1 nucleotide sequence is SEQID NO: 8. The human derived frzb-1 protein is illustrated by SEQ ID NO:9, and the human frzb-1 nucleotide sequence is SEQ ID NO: 10.

[0013] Frzb-1 is an antagonist of Wnts in vivo, and thus is believed tofind utility as a tumor suppressor gene, since overexpressed Wntproteins cause cancer. Frzb-1 may also be a useful vehicle forsolubilization and therapeutic delivery of wnt proteins complexed withit.

[0014] The final cDNA isolated containing a signal sequence results in apeptide designated Paraxial Protocadherin (PAPC). The cDNA for PAPC is adivergent member of the cadherin multigene family. PAPC is most relatedto protocadherin 43 reported by Sano et al., The EMBO J., 12, pp.2249-2256, 1993. As shown in SEQ ID NO: 5, the PAPC gene encodes atransmembrane protein of 896 amino acids, of which 187 are part of anintracellular domain. PAPC is a cell adhesion molecule, andmicroinjection of PAPC mRNA constructs into Xenopus embryos suggest thatPAPC acts as a molecule involved in mesoderm differentiation. A solubleform of the PAPC extracellular domain is able to block muscle andmesoderm formation in Xenopus embryos. The nucleotide sequence encodingXenopus PAPC is provided in SEQ ID NO: 6.

[0015] Cerberus, frzb-1, or PAPC or fragments thereof (which also may besynthesized by in vitro methods) may be fused (by recombinant expressionor in vitro covalent methods) to an immunogenic polypeptide and this, inturn, may be used to immunize an animal in order to raise antibodiesagainst the novel proteins. Antibodies are recoverable from the serum ofimmunized animals. Alternatively, monoclonal antibodies may be preparedfrom cells from the immunized animal in conventional fashion.Immobilized antibodies are useful particularly in the diagnosis (invitro or in vivo) or purification of cerberus, frzb-1, or PAPC.

[0016] Substitutional, deletional, or insertional mutants of the novelpolypeptides may be prepared by in vitro or recombinant methods andscreened for immuno-crossreactivity with cerberus, frzb-1, or PAPC andfor cerberus antagonist or agonist activity.

[0017] Cerberus or frzb-1 also may be derivatized in vitro in order toprepare immobilized and labelled proteins, particularly for purposes ofdiagnosis of insufficiencies thereof, or for affinity purification ofantibodies thereto.

[0018] Among applications for the novel proteins are tissue replacementtherapy and, because frzb-1 is an antagonist of Wnt signaling, tumorsuppression therapies. The cerberus receptor may define a novelsignalling pathway. In addition, frzb-1 could permit the isolation ofnovel members of the Wnt family of growth factors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 illustrates the amino acid sequence (SEQ ID NO: 1) of theFIG. 2 cDNA clone for cerberus;

[0020]FIG. 2 illustrates a cDNA clone (SEQ ID NO: 2) for cerberusderived from Xenopus. Sense strand is on top (5′ to 3′ direction) andthe antisense strand on the bottom line (in the opposite direction);

[0021]FIGS. 3 and 4 show the amino acid and nucleotide sequence,respectively, of full-length frzb-1 from Xenopus (SEQ ID NOS: 3 and 4);

[0022]FIGS. 5 and 6 show the amino acid and nucleotide sequence,respectively, of full-length PAPC from Xenopus (SEQ ID NOS: 5 and 6);

[0023]FIGS. 7 and 8 show the amino acid and nucleotide sequence,respectively, of full-length frzb-1 from mouse (SEQ ID NOS: 7 and 8);and

[0024]FIGS. 9 and 10 show the amino acid and nucleotide sequence,respectively, of full-length frzb-1 from human (SEQ ID NOS: 9 and 10).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Among the several novel proteins and their nucleotide sequencesdescribed herein, is a novel endodermal, cardiac, and neural inducingfactor in vertebrates that we have named “cerberus.” When referring tocerberus, the present invention also contemplates the use of fragments,derivatives, agonists, or antagonists of cerberus molecules. Becausecerberus has no homology to any reported growth factors, it is proposedto be the founding member of a novel family of growth factors withpotent biological activities, which may be isolated using SEQ ID NO: 2.

[0026] The amphibian organizer consists of several cell populations withregion-specific inducing activities. On the basis of morphogeneticmovements, three very different cell populations can be distinguished inthe organizer. First, cells with crawling migration movements involute,fanning out to form the prechordal plate. Second, cells involute throughthe dorsal lip driven by convergence and extension movements, givingrise to the notochord of the trunk. Third, involution ceases and thecontinuation of mediolateral intercalation movements leads to posteriorextension movements and to the formation of the tail notochord and ofthe chordoneural hinge. The three cell populations correspond to thehead, trunk, and tail organizers, respectively.

[0027] The cerberus gene is expressed at the right time and place toparticipate in cell signalling by Spemann's organizer. Specifically,cerberus is expressed in the head organizing region that consists ofcrawling-migrating cells. The cerberus expressing region corresponds tothe prospective foregut, including the liver and pancreas anlage, andthe heart mesoderm. Cerberus expression is activated by chordin, noggin,and organizer-specific homeobox genes.

[0028] Our studies were conducted in early embryos of the frog Xenopuslaevis. The frog embryo is well suited to experiments, particularlyexperiments pertaining to generating and maintaining regionaldifferences within the embryo for determining roles in tissuedifferentiation. It is easy to culture embryos with access to theembryos even at very early stages of development (preceding and duringthe formation of body pattern and differentiation) and the embryos arelarge. The initial work with noggin and chordin also had been in Xenopusembryos, and, as predicted, was highly conserved among vertebrates.Predictions based on work with Xenopus as to corresponding human nogginwere proven true and the ability to clone the gene for human noggin wasreadily accomplished. (See the description of Xenopus work and cloninginformation in PCT application, published Mar. 17, 1994, WO 9 405 800,and the subsequent human cloning based thereon in the PCT application,also published Mar. 17, 1994, as WO 9 405 791.)

CLONING

[0029] The cloning of cerberus, frzb-1, and PAPC resulted from acomprehensive screen for cDNAs enriched in Spemann's organizer.Subtractive differential screening was performed as follows. In brief,poly A⁺ RNA was isolated from 300 dorsal lip and ventral marginal zone(VMZ) explants at stage 10½. After first strand cDNA synthesisapproximately 70-80% of common sequences were removed by substractionwith biotinylated VMZ poly A⁺ RNA prepared from 1500 ventral gastrulahalves. For differential screening, duplicate filters (2000 plaques per15 cm plate, a total of 80,000 clones screened) of an unamplifiedoriented dorsal lip library were hybridized with radiolabeled dorsal lipor VMZ cDNA. Putative organizer-specific clones were isolated, groupedby sequence analysis from the 5′ end and whole-mount in situhybridization, and subsequently classified into known and newdorsal-specific genes. Rescreening of the library (100,000 independentphages) with a cerberus probe resulted in the isolation of 45 additionalclones, 31 of which had similar size as the longest one of the 11original clones indicating that they were presumably full-length cDNAs.The longest cDNAs for cerberus, frzb-1, and PAPC were completelysequenced.

[0030] To explore the molecular complexity of Spemann's organizer weperformed a comprehensive differential screen for dorsal-specific cDNAs.The method was designed to identify abundant cDNAs without bias as totheir function. As shown in Table 1, five previously known cDNAs andfive new ones were isolated, of which three (expressed as cerberus,frzb-1, and PAPC, respectively) had secretory signal sequences. TABLE 1Gene Product No. of Isolates Previously Known Genes Chordin novelsecreted protein 70 Goosecoid homeobox gene 3 Pintallavis/XFKH-1forkhead/transcription factor 2 Xnot-2 homeobox gene 1 Xlim-1 homeoboxgene 1 New Genes Cerberus novel secreted protein 11 PAPCcadherin-like/transmembrane 2 Frzb-1 novel secreted protein 1 Sox-2sry/transcription factor 1 Fkh-like forkhead/transcription factor 1

[0031] The most abundant dorsal-specific cDNA was chordin (chd), with 70independent isolates. The second most abundant cDNA was isolated 11times and named cerberus (after a mythological guardian dog withmultiple heads). The cerberus cDNA encodes a putative secretedpolypeptide of 270 amino acids, with an amino terminal hydrophobicsignal sequence and a carboxy terminal cysteine-rich region (FIG. 1).Cerberus is expressed specifically in the head organizer region of theXenopus embryo, including the future foregut.

[0032] An abundant mRNA found in the dorsal region of the Xenopusgastrula encodes the novel putative secreted protein we have designatedas cerberus. Cerberus mRNA has potent inducing activity in Xenopusembryos, leading to the formation of ectopic heads. Unlike otherorganizer-specific factors, cerberus does not dorsalize mesoderm and isinstead an inhibitor of trunk-tail mesoderm. Cerberus is expressed inthe anterior-most domain of the gastrula including the leading edge ofthe deep layer of the dorsal lip a region that, as shown here, givesrise to foregut and midgut endoderm. Cerberus promotes the formation ofcement gland, olfactory placodes, cyclopic eyes, forebrain, andduplicated heart and liver (a foregut derivative). Because the pancreasis also derived from this foregut region, it is likely that cerberusinduces pancreas in addition to liver. The expression pattern andinducing activities of cerberus suggest a role for a previouslyneglected region of the embryo, the prospective foregut endoderm, in theinduction of the anterior head region of the embryo.

[0033] Turning to FIG. 1, Xenopus cerberus encodes a putative secretedprotein transiently expressed during embryogenesis and the deduced aminoacid sequence of Xenopus cerberus is shown. The signal peptide sequenceand the nine cysteine residues in the carboxy-terminus are indicated inbold. Potential N-linked glycosylation sites are underlined. In databasesearches the cerberus protein showed limited similarity only to themammalian Dan protein, a possible tumor suppressor proposed to be aDNA-binding protein.

[0034] Cerberus appears to be a pioneer protein, as its amino acidsequence and the spacing of its 9 cysteine residues were notsignificantly similar to other proteins in the databases (NCBI-Gen Bankrelease 93.0). We conclude that the second most abundant dorsal-specificcDNA encodes a novel putative secreted factor, which should be thefounding member of a novel family of growth factors active in celldifferentiation.

[0035] Cerberus Demarcates an Anterior Organizer Domain. Cerberus mRNAis expressed at low levels in the unfertilized egg, and zygotictranscripts start accumulating at early gastrula. Expression continuesduring gastrula and early neurula, rapidly declining during neurulation.Importantly, cerberus expression starts about one hour after that ofchd, suggesting that cerberus could act downstream of the chd signal.

[0036] Whole-mount in situ hybridizations reveal that expression startsin the yolky endomesodermal cells located in the deep layer of theorganizer. The cerberus domain includes the leading edge of the mostanterior organizer cells and extends into the lateral mesoderm. Theleading edge gives rise to liver, pancreas, and foregut in its midline,and the more lateral region gives rise to heart mesoderm at later stagesof development.

[0037]FIG. 2 sets out the sequence of a full length Xenopus cDNA forcerberus.

[0038] This entirely new molecule has demonstrated physiologicalproperties that should prove useful in therapeutic, diagnostic, andclinical applications that require regeneration, differentiation, orrepair of tissues, such wound repair, neuronal regenerational ortransplantation, supplementation of heart muscle differentiation,differentiation of pancreas and liver, and other applications in whichcell differentiation processes are to be induced.

[0039] The second, novel, secreted protein we have discovered is called“frzb-1,” which was shown to be a secreted protein in Xenopus oocytemicroinjection experiments. Thus it provides a natural soluble form ofthe related extracellular domains of Drosophila and vertebrate frizzledproteins. we propose that the latter proteins could be converted intoactive soluble forms by introducing a stop codon before the firsttransmembrane domain. We have noted that the cysteine-rich region offrzb-1 and frizzled contains some overall structural homology with Wntproteins using the Profile Search homology program (Gribskov, Meth.Enzymol., 183, pp. 146-159, 1990). This had raised the interestingpossibility that frzb-1 could interact directly with Wnt growth factorsin the extracellular space. This was because we had found that whenmicroinjected into Xenopus embryos, frzb-1 constructs have moderatedorsalizing activity, leading to the formation of embryos with enlargedbrain and head, and shortened truck. Somatic muscle differentiation,which requires Xwnt-8, was inhibited. In the case of frzb-1, anattractive hypothesis, suggested by the structural homologies, was thatit may act as an inhibitor of Wnt-8, a growth factor that hasventralizing activity in the Xenopus embryo (Christian and Moon, GenesDev., 7, pp. 13-28, 1993). We have shown that frzb-1 can interact withXwnt-8 and Wnt-1, and it is expected that it could also interact withother members of the wnt family of growth factors, of which at least 15members exist in mammals. In addition, a possible interaction with Wntswas suggested by the recent discovery that dishevelled, a gene actingdownstream of wingless, has strong genetic interaction with frizzledmutants in Drosophila (Krasnow et al., Development, 121, pp. 4095-4102,1995). This possibility has been explored in depth (Leyns et al., Cell,88, pp. 747-756, Mar. 21, 1997), because a soluble antagonist of the Wntfamily of proteins is expected to be of great therapeutic value.Examples 1 and 2 illustrate tests that show antagonism of Xwnt-8 bybinding to frzb-1.

[0040] Vertebrate homologues of Frizzled have been isolated and they tooare anchored to the cell membrane by seven membrane spanning domains(Wang et al., J. Biol. Chem., 271, pp. 4468-4476, 1996). Frzb-1 differsfrom the frizzled proteins in that it is an entirely soluble, diffusiblesecreted protein and therefore suitable as a therapeutic agent. Thenucleotide sequence that when expressed results in frzb-1 protein isillustrated by SEQ ID NO: 4.

[0041] SEQ ID NO: 4 corresponds to the Xenopus homolog, but by using itin BLAST searches (and by cloning mouse frzb-1) we had been able toassemble the sequence of the entire mature human frzb-1 protein, SEQ IDNO: 9. Indeed, human frzb-1 is encoded in six expressed sequence tags(ESTs) available in Genebank. The human frzb-1 sequence can be assembledby overlapping in the 5′ to 3′ direction the ESTs with the followingaccession numbers in Genebank: H18848, R63748, W38677, W44760, H38379,and N71244. No function had yet been assigned to these EST sequences,but we believe and thus propose here that human frzb-1 will have similarfunctions in cell differentiation to those described above for Xenopusfrzb-1. The nucleotide sequence of human frzb-1 is shown in SEQ ID NO:10. The mouse frzb-1 protein and nucleotide sequences are provided bySEQ ID NOS: 7 and 8, respectively.

[0042] In particular, we believe that frzb-1 will prove useful in genetherapy of human cancer cells. In this rapidly developing field, oneapproach is to introduce vectors expressing anti-sense sequences toblock expression of dominant ocogenes and growth factor receptors.Another approach is to produce episomal vectors that will replicate inhuman cells in a controlled fashion without transforming the cells. Foran example of the latter (an episomal expression vector system for humangene therapy), reference is made to U.S. Pat. No. 5,624,820, issued Apr.29, 1997, inventor Cooper.

[0043] Gene therapy now includes uses of human tumor suppression genes.For example, U.S. Pat. No. 5,491,064, issued Feb. 13, 1996, discloses atumor suppression gene localized on chromosome 11 and described aspotentially useful for gene therapy in cancers deleted or altered intheir expression of that gene. Frzb-1 maps to chromosome 2q31-33 andloss of one copy of the 2q31-33 and loss of one copy of the 2q arm hasbeen observed with high incidence in lung carcinomas, colo-rectalcarcinomas, and neuroblastomas, which has lead to the proposal that the2q arm carries a tumor suppressor gene. We expect frzb to be a tumorsuppressor gene, and thus to be useful in tumor suppressionapplications.

[0044] A number of applications for cerberus and frzb-1 are suggestedfrom their pharmacological (biological activity) properties.

[0045] For example, the cerberus and frzb-1 cDNAs should be useful as adiagnostic tool (such as through use of antibodies in assays forproteins in cell lines or use of oligonucleotides as primers in a PCRtest to amplify those with sequence similarities to the oligonucleotideprimer, and to determine how much of the novel protein is present).

[0046] Cerberus, of course, might act upon its target cells via its ownreceptor. Cerberus, therefore, provides the key to isolate thisreceptor. Since many receptors mutate to cellular oncogenes, thecerberus receptor should prove useful as a diagnostic probe for certaintumor types. Thus, when one views cerberus as ligand in complexes, thencomplexes in accordance with the invention include antibody bound tocerberus, antibody bound to peptides derived from cerberus, cerberusbound to its receptor, or peptides derived from cerberus bound to itsreceptor or other factors. Mutant forms of cerberus, which are eithermore potent agonists or antagonists, are believed to be clinicallyuseful. Such complexes of cerberus and its binding protein partners willfind uses in a number of applications.

[0047] Practice of this invention includes use of an oligonucleotideconstruct comprising a sequence coding for cerberus or frzb-1 and for apromoter sequence operatively linked in a mammalian or a viralexpression vector. Expression and cloning vectors contain a nucleotidesequence that enables the vector to replicate in one or more selectedhost cells. Generally, in cloning vectors this sequence is one thatenables the vector to replicate independently of the host chromosomes,and includes origins of replication or autonomously replicatingsequences. The well-known plasmid pBR322 is suitable for most gramnegative bacteria, the 2μ plasmid origin for yeast and various viralorigins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloningvectors in mammalian cells.

[0048] Expression and cloning vectors should contain a selection gene,also termed a selectable marker. Typically, this is a gene that encodesa protein necessary for the survival or growth of a host celltransformed with the vector. The presence of this gene ensures that anyhost cell which deletes the vector will not obtain an advantage ingrowth or reproduction over transformed hosts. Typical selection genesencode proteins that (a) confer resistance to antibiotics or othertoxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, (b)complement auxotrophic deficiencies.

[0049] Examples of suitable selectable markers for mammalian cells aredihydrofolate reductase (DHFR) or thymidine kinase. Such markers enablethe identification of cells which were competent to take up the cerberusnucleic acid. The mammalian cell transformants are placed underselection pressure which only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed.Amplification is the process by which genes in greater demand for theproduction of a protein critical for growth are reiterated in tandemwithin the chromosomes of successive generations of recombinant cells.Increased quantities of cerberus or frzb-1 can therefor be synthesizedfrom the amplified DNA.

[0050] For example, cells transformed with the DHFR selection gene arefirst identified by culturing all of the transformants in a culturemedium which contains methotrexate (Mtx), a competitive antagonist ofDHFR. An appropriate host cell in this case is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub and Chasin, Proc. Nat. Acac. Sci., 77, 4216 (1980).The transformed cells then are exposed to increased levels of Mtx. Thisleads to the synthesis of multiple copies of the DHFR gene and,concomitantly, multiple copies of other DNA comprising the expressionvectors, such as the DNA encoding cerberus or frzb-1. Alternatively,host cells transformed by an expression vector comprising DNA sequencesencoding cerberus or frzb-1 and aminoglycoside 3′ phosphotransferase(APH) protein can be selected by cell growth in medium containing anaminoglycosidic antibiotic such as kanamycin or neomycin or G418.Because eukaryotic cells do not normally express an endogenous APHactivity, genes encoding APH protein, commonly referred to as neoresistant genes, may be used as dominant selectable markers in a widerange of eukaryotic host cells, by which cells transformed by the vectorcan readily be identified.

[0051] Expression vectors, unlike cloning vectors, should contain apromoter which is recognized by the host organism and is operably linkedto the cerberus nucleic acid. Promoters are untranslated sequenceslocated upstream from the start codon of a structural gene (generallywithin about 100 to 1000 bp) that control the transcription andtranslation of nucleic acid under their control. They typically fallinto two classes, inducible and constitutive. Inducible promoters arepromoters that initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, e.g. thepresence or absence of a nutrient or a change in temperature. At thistime a large number of promoters recognized by a variety of potentialhost cells are well known. These promoters can be operably linked tocerberus encoding DNA by removing them from their gene of origin byrestriction enzyme digestion, followed by insertion 5′ to the startcodon for cerberus or frzb-1.

[0052] Nucleic acid is operably linked when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein which participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,operably linked means that the DNA sequences being linked are contiguousand, in the case of a secretory leader, contiguous and in reading phase.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exit then synthetic oligonucleotide adapters orlinkers are used in accord with conventional practice.

[0053] Transcription of the protein-encoding DNA in mammalian host cellsis controlled by promoters obtained from the genomes of viruses such aspolyoma, cytomegalo-virus, adenovirus, retroviruses, hepatitis-B virus,and most preferably Simian Virus 40 (SV40), or from heterologousmammalian promoters, e.g. the actin promoter. Of course, promoters fromthe host cell or related species also are useful herein.

[0054] Cerberus and frzb-1 are clearly useful as a component of culturemedia for use in culturing cells, such as endodermal, cardiac, and nervecells, in vitro. We believe cerberus and frzb-1 will find uses as agentsfor enhancing the survival or inducing the growth of liver, pancreas,heart, and nerve cells, such as in tissue replacement therapy.

[0055] The final cDNA isolated containing a signal sequence results in apeptide designated Paraxial Protocadherin (PAPC). The cDNA for PAPC is adivergent member of the cadherin multigene family. PAPC is most relatedto protocadherin 43 reported by Sano et al., The EMBO J., 12, pp.2249-2256, 1993. As shown in SEQ ID NO: 5, the PAPC gene encodes atransmembrane protein of 896 amino acids, of which 187 are part of anintracellular domain. PAPC is a cell adhesion molecule, andmicroinjection of PAPC mRNA constructs into Xenopus embryos suggest thatPAPC acts in mesoderm differentiation. The nucleotide sequence encodingXenopus PAPC is provided in SEQ ID NO: 6.

[0056] Therapeutic formulations of the novel proteins may be preparedfor storage by mixing the polypeptides having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers, in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;anti-oxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin or immunoglobulins. Other components can include glycine,blutamine, asparagine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, Pluronics or PEG.

[0057] Polyclonal antibodies to the novel proteins generally are raisedin animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of cerberus or frzb-1 and an adjuvant. It may be useful toconjugate these proteins or a fragment containing the target amino acidsequence to a protein which is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR.

[0058] Animals can be immunized against the immunogenic conjugates orderivatives by combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freund's complete adjuvant and injectingthe solution intradermally in multiple sites. One month later theanimals are boosted with 1/5 to 1/10 the original amount of conjugate inFruend's complete adjuvant by subcutaneous injection at multiple sites.Seven to 14 days later animals are bled and the serum is assayed foranti-cerberus titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the samecerberus or frzb-1 polypeptide, but conjugated to a different proteinand/or through a different cross-linking agent. Conjugates also can bemade in recombinant cell culture as protein fusions. Also, aggregatingagents such as alum are used to enhance the immune response.

[0059] Monoclonal antibodies are prepared by recovering spleen cellsfrom immunized animals and immortalizing the cells in conventionalfashion, e.g. by fusion with myeloma cells or by EB virus transformationand screening for clones expressing the desired antibody.

[0060] Antibodies are useful in diagnostic assays for cerberus, frzb-1,or PAPC or their antibodies and to identify family members. In oneembodiment of a receptor binding assay, an antibody composition whichbinds to all of a selected plurality of members of the cerberus familyis immobilized on an insoluble matrix, the test sample is contacted withthe immobilized antibody composition in order to adsorb all cerberusfamily members, and then the immobilized family members are contactedwith a plurality of antibodies specific for each member, each of theantibodies being individually identifiable as specific for apredetermined family member, as by unique labels such as discretefluorophores or the like. By determining the presence and/or amount ofeach unique label, the relative proportion and amount of each familymember can be determined.

[0061] The antibodies also are useful for the affinity purification ofthe novel proteins from recombinant cell culture or natural sources.Antibodies that do not detectably cross-react with other growth factorscan be used to purify the proteins free from these other family members.

EXAMPLE 1 Frzb-1 Antagonizes Xwnt-8 Non-Cell Autonomously

[0062] To test whether frzb-1 can antagonize secondary axes caused byXwnt-8 after secretion by injected cells, an experimental design wasused. Thus, frzb-1 mRNA was injected into each of the four animalblastomeres of eight-cell embryos, and subsequently, a single injectionof Xwnt-8 mRNA was given to a vegetal-ventral blastomere at the 16-32cell stage. In two independent experiments, we found that injection offrzb-1 alone (n=13) caused mild dorsalization with enlargement of thecement gland in all embryos and that injection of Xwnt-8 alone (n=53)lead to induction of complete secondary axes in 67% of the embryos.However, injection of frzb-1 into animal caps abolished the formation ofcomplete axes induced by Xwnt-8 (n=27), leaving only a residual 14% ofembryos with very weak secondary axes. The double-injected embryosretained the enlarged cement gland phenotype caused by injection offrzb-1 mRNA alone. Because both mRNAs encode secreted proteins and weremicroinjected into different cells, we conclude that the antagonisticeffects of frzb-1 and Xwnt-8 took place in the extracellular space afterthese proteins were secreted.

EXAMPLE 2 Membrane-Anchored Wnt-1 Confers Frzb-1 Binding

[0063] To investigate a possible interaction between frzb-1 and Wnts,the first step was to insert an HA epitope tag into a Xenopus frzb-1construct driven by the CMV (cytomegalovirus) promoter. Frzb1-HA wastested in mRNA microinjection assays in Xenopus embryos and found to bebiologically active. Conditioned medium from transiently transfectedcells contained up to 10 μg/ml of Frzb1-HA (quantitated on Western blotsusing an HA-tagged protein standard).

[0064] Transient transfection of 293 cells has been instrumental indemonstrating interactions between wingless and frizzled proteins. Wetherefore took advantage of constructs in which Wnt-1 was fused at theamino terminus of CD8, generating a transmembrane protein containingbiologically active Wnt-1 exposed to the extracellular compartment. AWnt1CD8 cDNA construct (a generous gift of Dr. H. Varmus , NIH) wassubcloned into the pcDNA (Invitrogen) vector and transfected into 293cells. After incubation with Frzb1-HA-conditioned medium (overnight at37° C.), intensely labeled cells were observed by immunofluorescence. Asa negative control, a construct containing 120 amino acids of Xenopuschordin, an unrelated secreted protein was used. Transfection of thisconstruct produced background binding of Frzb1-HA to the extracellularmatrix, both uniform and punctate. Cotransfection of Wnt1CD8 withpcDNA-LacZ showed that transfected cells stained positively for Frzb1-HAand LacZ. Since Wnt1CD8 contains the entire CD8 molecule, a CD8 cDNA wasused as an additional negative control. After transfection with Lacz andfull-length CE8, Frzb1-HA failed to bind to the transfected cells.Although most of our experiments were carried out at 37° C.,Frzb1-HA-conditioned medium also stained Wnt1CD8-transfected cells afterincubation at 4° C. for 2 hours.

[0065] Attempts to biochemically quantitate the binding of Frzb-1 toWnt1CD8-transfected cells were unsuccessful due to high backgroundbinding to control cultures, presumably due to binding to theextracellular matrix. Thus, we were unable to estimate a K_(D) for theaffinity of the Frzb-1/Wnt-1 interaction. However, when serial dilutionsof conditioned medium containing Frzb1-HA were performed (ranging from2.5×10⁻⁷ to 1.25×10⁻¹⁰ M), staining of Wnt1CD8-transfected cells wasfound at all concentrations.

[0066] Although we have been unable to provide biochemical evidence fordirect binding between Wnts and frzb-1, this cell biological assayindicates that Frzb1-HA can bind, directly or indirectly, to Wnt-1 onthe cell membrane in the 10⁻¹⁰ M range.

[0067] It is to be understood that while the invention has beendescribed above in conjunction with preferred specific embodiments, thedescription and examples are intended to illustrate and not limit thescope of the invention, which is defined by the scope of the appendedclaims.

1 10 1 270 PRT Xenopus 1 Met Leu Leu Asn Val Leu Arg Ile Cys Ile Ile ValCys Leu Val Asn 1 5 10 15 Asp Gly Ala Gly Lys His Ser Glu Gly Arg GluArg Thr Lys Thr Tyr 20 25 30 Ser Leu Asn Ser Arg Gly Tyr Phe Arg Lys GluArg Gly Ala Arg Arg 35 40 45 Ser Lys Ile Leu Leu Val Asn Thr Lys Gly LeuAsp Glu Pro His Ile 50 55 60 Gly His Gly Asp Phe Gly Leu Val Ala Glu LeuPhe Asp Ser Thr Arg 65 70 75 80 Thr His Thr Asn Arg Lys Glu Pro Asp MetAsn Lys Val Lys Leu Phe 85 90 95 Ser Thr Val Ala His Gly Asn Lys Ser AlaArg Arg Lys Ala Tyr Asn 100 105 110 Gly Ser Arg Arg Asn Ile Phe Ser ArgArg Ser Phe Asp Lys Arg Asn 115 120 125 Thr Glu Val Thr Glu Lys Pro GlyAla Lys Met Phe Trp Asn Asn Phe 130 135 140 Leu Val Lys Met Asn Gly AlaPro Gln Asn Thr Ser His Gly Ser Lys 145 150 155 160 Ala Gln Glu Ile MetLys Glu Ala Cys Lys Thr Leu Pro Phe Thr Gln 165 170 175 Asn Ile Val HisGlu Asn Cys Asp Arg Met Val Ile Gln Asn Asn Leu 180 185 190 Cys Phe GlyLys Cys Ile Ser Leu His Val Pro Asn Gln Gln Asp Arg 195 200 205 Arg AsnThr Cys Ser His Cys Leu Pro Ser Lys Phe Thr Leu Asn His 210 215 220 LeuThr Leu Asn Cys Thr Gly Ser Lys Asn Val Val Lys Val Val Met 225 230 235240 Met Val Glu Glu Cys Thr Cys Glu Ala His Lys Ser Asn Phe His Gln 245250 255 Thr Ala Gln Phe Asn Met Asp Thr Ser Thr Thr Leu His His 260 265270 2 1338 DNA Xenopus 2 gaattcccag caagtcgctc agaaacactg cagggtctagatatcataca atgttactaa 60 atgtactcag gatctgtatt atcgtctgcc ttgtgaatgatggagcagga aaacactcag 120 aaggacgaga aaggacaaaa acatattcac ttaacagcagaggttacttc agaaaagaaa 180 gaggagcacg taggagcaag attctgctgg tgaatactaaaggtcttgat gaaccccaca 240 ttgggcatgg tgattttcgc ttagtagctg aactatttgattccaccaga acacatacaa 300 acagaaaaga gccagacatg aacaaagtca agcttttctcaacagttgcc catggaaaca 360 aaagtgcaag aagaaaagct tacaatggtt ctagaaggaatatttttcct cgccgttctt 420 ttgataaaag aaatacagag gttactgaaa agcctggtgccaagatgttc tggaacaatt 480 ttttggttaa aatgaatgga gccccacaga atacaagccatggcagtaaa gcacaggaaa 540 taatgaaaga agcttgcaaa accttgtttt tcactcagaatattgtacat gaaaactgtg 600 acaggatggt gatacagaac aatctgtgct ttggtaaatgcatctctctc catgttccaa 660 atcagcaaga tcgacgaaat acttgttccc attgcttgccgtccaaattt accctgaacc 720 acctgacgct gaattgtact ggatctaaga atgtagtaaaggttgtcatg atggtagagg 780 aatgcacgtg tgaagctcat aagagcaact tccaccaaactgcacagttt aacatggata 840 catctactac cctgcaccat taaaggactg ccatacagtatggaaatgcc cttttgttgg 900 aatatttgtt acatactatg catctaaagc attatgttgccttctatttc atataaccac 960 atggaataag gattgtatga attataatta acaaatggcattttgtgtaa catgcaagat 1020 ctctgttcca tcagttgcaa gataaaaggc aatatttgtttgactttttt tctacaaaat 1080 gaatacccaa atatatgata agataatggg gtcaaaactgttaaggggta atgtaataat 1140 agggactaag tttgcccagg agcagtgacc cataacaaccaatcagcagg tatgatttac 1200 tggtcacctg tttaaaagca aacatcttat tggttgctatgggttactgc ttctgggcaa 1260 aatgtgtgcc tcataggggg gttagtgtgt tgtgtactgaataaattgta tttatttcat 1320 tgttacaaaa aaaaaaaa 1338 3 318 PRT Xenopusfrazzled 3 Met Ser Arg Thr Arg Lys Val Asp Ser Leu Leu Leu Leu Ala IlePro 1 5 10 15 Gly Leu Ala Leu Leu Leu Leu Pro Asn Ala Tyr Cys Ala SerCys Glu 20 25 30 Pro Val Arg Ile Pro Met Cys Lys Ser Met Pro Trp Asn MetThr Lys 35 40 45 Met Pro Asn His Leu His His Ser Thr Gln Ala Asn Ala IleLeu Ala 50 55 60 Ile Glu Gln Phe Glu Gly Leu Leu Thr Thr Glu Cys Ser GlnAsp Leu 65 70 75 80 Leu Phe Phe Leu Cys Ala Met Tyr Ala Pro Ile Cys ThrIle Asp Phe 85 90 95 Gln His Glu Pro Ile Lys Pro Cys Lys Ser Val Cys GluArg Ala Arg 100 105 110 Ala Gly Cys Glu Pro Ile Leu Ile Lys Tyr Arg HisThr Trp Pro Glu 115 120 125 Ser Leu Ala Cys Glu Glu Leu Pro Val Tyr AspArg Gly Val Cys Ile 130 135 140 Ser Pro Glu Ala Ile Val Thr Val Glu GlnGly Thr Asp Ser Met Pro 145 150 155 160 Asp Phe Ser Met Asp Ser Asn AsnGly Asn Cys Gly Ser Gly Arg Glu 165 170 175 His Cys Lys Cys Lys Pro MetLys Ala Thr Gln Lys Thr Tyr Leu Lys 180 185 190 Asn Asn Tyr Asn Tyr ValIle Arg Ala Lys Val Lys Glu Val Lys Val 195 200 205 Lys Cys His Asp AlaThr Ala Ile Val Glu Val Lys Glu Ile Leu Lys 210 215 220 Ser Ser Leu ValAsn Ile Pro Lys Asp Thr Val Thr Leu Tyr Thr Asn 225 230 235 240 Ser GlyCys Leu Cys Pro Gln Leu Val Ala Asn Glu Glu Tyr Ile Ile 245 250 255 MetGly Tyr Glu Asp Lys Glu Arg Thr Arg Leu Leu Leu Val Glu Gly 260 265 270Ser Leu Ala Glu Lys Trp Arg Asp Arg Leu Ala Lys Lys Val Lys Arg 275 280285 Trp Asp Gln Lys Leu Arg Arg Pro Arg Lys Ser Lys Asp Pro Val Ala 290295 300 Pro Ile Pro Asn Lys Asn Ser Asn Ser Arg Gln Ala Arg Ser 305 310315 4 1875 DNA Xenopus frazzled 4 gaattccctt tcacacagga ctcctggcagaggtgaatgg ttagccctat ggatttggtt 60 tgttgatttt gacacatgat tgattgctttcagataggat tgaaggactt ggatttttat 120 ctaattctgc acttttaaat tatctgagtaattgttcatt ttgtattgga tgggactaaa 180 gataaactta actccttgct tttgacttgcccataaacta taaggtgggg tgagttgtag 240 ttgcttttac atgtgcccag attttccctgtattccctgt attccctcta aagtaagcct 300 acacatacag gttgggcaga ataacaatgtctcgaacaag gaaagtggac tcattactgc 360 tactggccat acctggactg gcgcttctcttattacccaa tgcttactgt gcttcgtgtg 420 agcctgtgcg gatccccatg tgcaaatctatgccatggaa catgaccaag atgcccaacc 480 atctccacca cagcactcaa gccaatgccatcctggcaat tgaacagttt gaaggtttgc 540 tgaccactga atgtagccag gaccttttgttctttctgtg tgccatgtat gcccccattt 600 gtaccatcga tttccagcat gaaccaattaagccttgcaa gtccgtgtgc gaaagggcca 660 gggccggctg tgagcccatt ctcataaagtaccggcacac ttggccagag agcctggcat 720 gtgaagagct gcccgtatat gacagaggagtctgcatctc cccagaggct atcgtcacag 780 tggaacaagg aacagattca atgccagacttctccatgga ttcaaacaat ggaaattgcg 840 gaagcggcag ggagcactgt aaatgcaagcccatgaaggc aacccaaaag acgtatctca 900 agaataatta caattatgta atcagagcaaaagtgaaaga ggtgaaagtg aaatgccacg 960 acgcaacagc aattgtggaa gtaaaggagattctcaagtc ttccctagtg aacattccta 1020 aagacacagt gacactgtac accaactcaggctgcttgtg cccccagctt gttgccaatg 1080 aggaatacat aattatgggc tatgaagacaaagagcgtac caggcttcta ctagtggaag 1140 gatccttggc cgaaaaatgg agagatcgtcttgctaagaa agtcaagcgc tgggatcaaa 1200 agcttcgacg tcccaggaaa agcaaagaccccgtggctcc aattcccaac aaaaacagca 1260 attccagaca agcgcgtagt tagactaacggaaaggtgta tggaaactct atggactttg 1320 aaactaagat ttgcattgtt ggaagagcaaaaaagaaatt gcactacagc acgttatatt 1380 ctattgttta ctacaagaag ctggtttagttgattgtagt tctcctttcc ttcttttttt 1440 ttataactat atttgcacgt gttcccaggcaattgtttta ttcaacttcc agtgacagag 1500 cagtgactga atgtctcagc ctaaagaagctcaattcatt tctgatcaac taatggtgac 1560 aagtgtttga tacttgggga aagtgaactaattgcaatgg taaatcagag aaaagttgac 1620 caatgttgct tttcctgtag atgaacaagtgagagatcac atttaaatga tgatcacttt 1680 ccatttaata ctttcagcag ttttagttagatgacatgta ggatgcacct aaatctaaat 1740 attttatcat aaatgaagag ctggtttagactgtatggtc actgttggga aggtaaatgc 1800 ctactttgtc aattctgttt taaaaattgcctaaataaat attaagtcct aaataaaaaa 1860 aaaaaaaaaa aaaaa 1875 5 896 PRTXenopus 5 Met Leu Leu Leu Phe Arg Ala Ile Pro Met Leu Leu Leu Gly LeuMet 1 5 10 15 Val Leu Gln Thr Asp Cys Glu Ile Ala Gln Tyr Tyr Ile AspGlu Glu 20 25 30 Glu Pro Pro Gly Thr Val Ile Ala Val Leu Ser Gln His SerIle Phe 35 40 45 Asn Thr Thr Asp Ile Pro Ala Thr Asn Phe Arg Leu Met LysGln Phe 50 55 60 Asn Asn Ser Leu Ile Gly Val Arg Glu Ser Asp Gly Gln LeuSer Ile 65 70 75 80 Met Glu Arg Ile Asp Arg Glu Gln Ile Cys Arg Gln SerLeu His Cys 85 90 95 Asn Leu Ala Leu Asp Val Val Ser Phe Ser Lys Gly HisPhe Lys Leu 100 105 110 Leu Asn Val Lys Val Glu Val Arg Asp Ile Asn AspHis Ser Pro His 115 120 125 Phe Pro Ser Glu Ile Met His Val Glu Val SerGlu Ser Ser Ser Val 130 135 140 Gly Thr Arg Ile Pro Leu Glu Ile Ala IleAsp Glu Asp Val Gly Ser 145 150 155 160 Asn Ser Ile Gln Asn Phe Gln IleSer Asn Asn Ser His Phe Ser Ile 165 170 175 Asp Val Leu Thr Arg Ala AspGly Val Lys Tyr Ala Asp Leu Val Leu 180 185 190 Met Arg Glu Leu Asp ArgGlu Ile Gln Pro Thr Tyr Ile Met Glu Leu 195 200 205 Leu Ala Met Asp GlyGly Val Pro Ser Leu Ser Gly Thr Ala Val Val 210 215 220 Asn Ile Arg ValLeu Asp Phe Asn Asp Asn Ser Pro Val Phe Glu Arg 225 230 235 240 Ser ThrIle Ala Val Asp Leu Val Glu Asp Ala Pro Leu Gly Tyr Leu 245 250 255 LeuLeu Glu Leu His Ala Thr Asp Asp Asp Glu Gly Val Asn Gly Glu 260 265 270Ile Val Tyr Gly Phe Ser Thr Leu Ala Ser Gln Glu Val Arg Gln Leu 275 280285 Phe Lys Ile Asn Ser Arg Thr Gly Ser Val Thr Leu Glu Gly Gln Val 290295 300 Asp Phe Glu Thr Lys Gln Thr Tyr Glu Phe Glu Val Gln Ala Gln Asp305 310 315 320 Leu Gly Pro Asn Pro Leu Thr Ala Thr Cys Lys Val Thr ValHis Ile 325 330 335 Leu Asp Val Asn Asp Asn Thr Pro Ala Ile Thr Ile ThrPro Leu Thr 340 345 350 Thr Val Asn Ala Gly Val Ala Tyr Ile Pro Glu ThrAla Thr Lys Glu 355 360 365 Asn Phe Ile Ala Leu Ile Ser Thr Thr Asp ArgAla Ser Gly Ser Asn 370 375 380 Gly Gln Val Arg Cys Thr Leu Tyr Gly HisGlu His Phe Lys Leu Gln 385 390 395 400 Gln Ala Tyr Glu Asp Ser Tyr MetIle Val Thr Thr Ser Thr Leu Asp 405 410 415 Arg Glu Asn Ile Ala Ala TyrSer Leu Thr Val Val Ala Glu Asp Leu 420 425 430 Gly Phe Pro Ser Leu LysThr Lys Lys Tyr Tyr Thr Val Lys Val Ser 435 440 445 Asp Glu Asn Asp AsnAla Pro Val Phe Ser Lys Pro Gln Tyr Glu Ala 450 455 460 Ser Ile Leu GluAsn Asn Ala Pro Gly Ser Tyr Ile Thr Thr Val Ile 465 470 475 480 Ala ArgAsp Ser Asp Ser Asp Gln Asn Gly Lys Val Asn Tyr Arg Leu 485 490 495 ValAsp Ala Lys Val Met Gly Gln Ser Leu Thr Thr Phe Val Ser Leu 500 505 510Asp Ala Asp Ser Gly Val Leu Arg Ala Val Arg Ser Leu Asp Tyr Glu 515 520525 Lys Leu Lys Gln Leu Asp Phe Glu Ile Glu Ala Ala Asp Asn Gly Ile 530535 540 Pro Gln Leu Ser Thr Arg Val Gln Leu Asn Leu Arg Ile Val Asp Gln545 550 555 560 Asn Asp Asn Cys Pro Val Ile Thr Asn Pro Leu Leu Asn AsnGly Ser 565 570 575 Gly Glu Val Leu Leu Pro Ile Ser Ala Pro Gln Asn TyrLeu Val Phe 580 585 590 Gln Leu Lys Ala Glu Asp Ser Asp Glu Gly His AsnSer Gln Leu Phe 595 600 605 Tyr Thr Ile Leu Arg Asp Pro Ser Arg Leu PheAla Ile Asn Lys Glu 610 615 620 Ser Gly Glu Val Phe Leu Lys Lys Gln LeuAsn Ser Asp His Ser Glu 625 630 635 640 Asp Leu Ser Ile Val Val Ala ValTyr Asp Leu Gly Arg Pro Ser Leu 645 650 655 Ser Thr Asn Ala Thr Val LysPhe Ile Leu Thr Asp Ser Phe Pro Ser 660 665 670 Asn Val Glu Val Val IleLeu Gln Pro Ser Ala Glu Glu Gln His Gln 675 680 685 Ile Asp Met Ser IleIle Phe Ile Ala Val Leu Ala Gly Gly Cys Ala 690 695 700 Leu Leu Leu LeuAla Ile Phe Phe Val Ala Cys Thr Cys Lys Lys Lys 705 710 715 720 Ala GlyGlu Phe Lys Gln Val Pro Glu Gln His Gly Thr Cys Asn Glu 725 730 735 GluArg Leu Leu Ser Thr Pro Ser Pro Gln Ser Val Ser Ser Ser Leu 740 745 750Ser Gln Ser Glu Ser Cys Gln Leu Ser Ile Asn Thr Glu Ser Glu Asn 755 760765 Cys Ser Val Ser Ser Asn Gln Glu Gln His Gln Gln Thr Gly Ile Lys 770775 780 His Ser Ile Ser Val Pro Ser Tyr His Thr Ser Gly Trp His Leu Asp785 790 795 800 Asn Cys Ala Met Ser Ile Ser Gly His Ser His Met Gly HisIle Ser 805 810 815 Thr Lys Val Gln Trp Ala Lys Glu Ile Val Thr Ser MetThr Val Thr 820 825 830 Leu Ile Leu Val Glu Asn Gln Lys Arg Arg Ala LeuSer Ser Gln Cys 835 840 845 Arg His Lys Pro Val Leu Asn Thr Gln Met AsnGln Gln Gly Ser Asp 850 855 860 Met Pro Ile Thr Ile Ser Ala Thr Glu SerThr Arg Val Gln Lys Met 865 870 875 880 Gly Thr Ala His Cys Asn Met LysArg Ala Ile Asp Cys Leu Thr Leu 885 890 895 6 3657 DNA Xenopus 6gaattcccag agatgaactc cttgagattg ttttaaatga ctgcaggtct ggaaggattc 60acattgccac actgtttcta ggcatgaaaa aactgcaagt ttcaactttg tttttggtgc 120aactttgatt cttcaagatg ctgcttctct tcagagccat tccaatgctg ctgttgggac 180tgatggtttt acaaacagac tgtgaaattg cccagtacta catagatgaa gaagaacccc 240ctggcactgt aattgcagtg ttgtcacaac actccatatt taacactaca gatatacctg 300caaccaattt ccgtctaatg aagcaattta ataattccct tatcggagtc cgtgagagtg 360atgggcagct gagcatcatg gagaggattg accgggagca aatctgcagg cagtcccttc 420actgcaacct ggctttggat gtggtcagct tttccaaagg acacttcaag cttctgaacg 480tgaaagtgga ggtgagagac attaatgacc atagccctca ctttcccagt gaaataatgc 540atgtggaggt gtctgaaagt tcctctgtgg gcaccaggat tcctttagaa attgcaatag 600atgaagatgt tgggtccaac tccatccaga actttcagat ctcaaataat agccacttca 660gcattgatgt gctaaccaga gcagatgggg tgaaatatgc agatttagtc ttaatgagag 720aactggacag ggaaatccag ccaacataca taatggagct actagcaatg gatgggggtg 780taccatcact atctggtact gcagtggtta acatccgagt cctggacttt aatgataaca 840gcccagtgtt tgagagaagc accattgctg tggacctagt agaggatgct cctctgggat 900accttttgtt ggagttacat gctactgacg atgatgaagg agtgaatgga gaaattgttt 960atggattcag cactttggca tctcaagagg tacgtcagct atttaaaatt aactccagaa 1020ctggcagtgt tactcttgaa ggccaagttg attttgagac caagcagact tacgaatttg 1080aggtacaagc ccaagatttg ggccccaacc cactgactgc tacttgtaaa gtaactgttc 1140atatacttga tgtaaatgat aataccccag ccatcactat tacccctctg actactgtaa 1200atgcaggagt tgcctatatt ccagaaacag ccacaaagga gaactttata gctctgatca 1260gcactactga cagagcctct ggatctaatg gacaagttcg ctgtactctt tatggacatg 1320agcactttaa actacagcaa gcttatgagg acagttacat gatagttacc acctctactt 1380tagacaggga aaacatagca gcgtactctt tgacagtagt tgcagaagac cttggcttcc 1440cctcattgaa gaccaaaaag tactacacag tcaaggttag tgatgagaat gacaatgcac 1500ctgtattttc taaaccccag tatgaagctt ctattctgga aaataatgct ccaggctctt 1560atataactac agtgatagcc agagactctg atagtgatca aaatggcaaa gtaaattaca 1620gacttgtgga tgcaaaagtg atgggccagt cactaacaac atttgtttct cttgatgcgg 1680actctggagt attgagagct gttaggtctt tagactatga aaaacttaaa caactggatt 1740ttgaaattga agctgcagac aatgggatcc ctcaactctc cactcgcgtt caactaaatc 1800tcagaatagt tgatcaaaat gataattgcc ctgtgataac taatcctctt cttaataatg 1860gctcgggtga agttctgctt cccatcagcg ctcctcaaaa ctatttagtt ttccagctca 1920aagccgagga ttcagatgaa gggcacaact cccagctgtt ctataccata ctgagagatc 1980caagcagatt gtttgccatt aacaaagaaa gtggtgaagt gttcctgaaa aaacaattaa 2040actctgacca ttcagaggac ttgagcatag tagttgcagt gtatgacttg ggaagacctt 2100cattatccac caatgctaca gttaaattca tcctcaccga ctcttttcct tctaacgttg 2160aagtcgttat tttgcaacca tctgcagaag agcagcacca gatcgatatg tccattatat 2220tcattgcagt gctggctggt ggttgtgctt tgctactttt ggccatcttt tttgtggcct 2280gtacttgtaa aaagaaagct ggtgaattta agcaggtacc tgaacaacac ggaacatgca 2340atgaagaacg cctgttaagc accccatctc cccagtcggt ctcttcttct ttgtctcagt 2400ctgagtcatg ccaactctcc atcaatactg aatctgagaa ttgcagcgtg tcctctaacc 2460aagagcagca tcagcaaaca ggcataaagc actccatctc tgtaccatct tatcacacat 2520ctggttggca cctggacaat tgtgcaatga gcataagtgg acattctcac atggggcaca 2580ttagtacaaa ggtacagtgg gcaaaggaga tagtgacttc aatgacagtg actctgatac 2640tagtggagaa tcacaaaaga agagcattga gcagccaatg caggcacaag ccagtgctca 2700atacacagat gaatcagcag ggttccgaca tgccgataac tatttcagcc accgaatcaa 2760caagggtcca gaaaatggga actgcacatt gcaatatgaa aagggctata gactgtctta 2820ctctgtagct cctgctcatt acaataccta ccatgcaaga atgcctaacc tgcacatacc 2880gaaccatacc cttagagacc cttattacca tatcaataat cctgttgcta atcggatgca 2940ggcggaatat gaaagagatt tagtcaacag aagtgcaacg ttatctccgc agagatcgtc 3000tagcagatac caagaattca attacagtcc gcagatatca agacagcttc atccttcaga 3060aattgctaca accttttaat cattaggcat gcaagtgaga atgcacaaag gcaagtgctt 3120tagcatgaaa gctaaatata tggagtctcc cctttccctc tgatggatgg ggggagacac 3180aggacagtgc ataaatatac agctgctttc tatttgcatt tcacttggga attttttgtt 3240ttttttacat atttattttt cctgaattga atgtgacatt gtcctgtcac ctaactagca 3300attaaatcca cagacctaca gtcaaatatt tgagggcccc tgaaacagca catcagtcag 3360gacctaaagt ggccttttta cttttagcag ctcctgggtc tgccctctgt gttaatcagc 3420ccctggtcaa gtcctgagta ggatcatggc gtttttatat gcatctcacc tactttggac 3480gtgatttaca cataatagga aacgcttggt ttcagtgaag tctgtgttgt atatattctg 3540ttatatacac gcattttgtg tttgtgtata tatttcaagt ccattcagat atgtgtatat 3600agtgcagacc ttgtaaaatt aatattctga tactttttcc tcaataaata tttaaat 3657 7323 PRT Mouse FRZB-1 7 Met Val Cys Cys Gly Pro Gly Arg Met Leu Leu GlyTrp Ala Gly Leu 1 5 10 15 Leu Val Leu Ala Ala Leu Cys Leu Leu Gln ValPro Gly Ala Gln Ala 20 25 30 Ala Ala Cys Glu Pro Val Arg Ile Pro Leu CysLys Ser Leu Pro Trp 35 40 45 Asn Met Thr Lys Met Pro Asn His Leu His HisSer Thr Gln Ala Asn 50 55 60 Ala Ile Leu Ala Met Glu Gln Phe Glu Gly LeuLeu Gly Thr His Cys 65 70 75 80 Ser Pro Asp Leu Leu Phe Phe Leu Cys AlaMet Tyr Ala Pro Ile Cys 85 90 95 Thr Ile Asp Phe Gln His Glu Pro Ile LysPro Cys Lys Ser Val Cys 100 105 110 Glu Arg Ala Arg Gln Gly Cys Glu ProIle Leu Ile Lys Tyr Arg His 115 120 125 Ser Trp Pro Glu Ser Leu Ala CysAsp Glu Leu Pro Val Tyr Asp Arg 130 135 140 Gly Val Cys Ile Ser Pro GluAla Ile Val Thr Ala Asp Gly Ala Asp 145 150 155 160 Phe Pro Met Asp SerSer Thr Gly His Cys Arg Gly Ala Ser Ser Glu 165 170 175 Arg Cys Lys CysLys Pro Val Arg Ala Thr Gln Lys Thr Tyr Phe Arg 180 185 190 Asn Asn TyrAsn Tyr Val Ile Arg Ala Lys Val Lys Glu Val Lys Met 195 200 205 Lys CysHis Asp Val Thr Ala Val Val Glu Val Lys Glu Ile Leu Lys 210 215 220 AlaSer Leu Val Asn Ile Pro Arg Asp Thr Val Asn Leu Tyr Thr Thr 225 230 235240 Ser Gly Cys Leu Cys Pro Pro Leu Thr Val Asn Glu Glu Tyr Val Ile 245250 255 Met Gly Tyr Glu Asp Glu Glu Arg Ser Arg Leu Leu Leu Val Glu Gly260 265 270 Ser Ile Ala Glu Lys Trp Lys Asp Arg Leu Gly Lys Lys Val LysArg 275 280 285 Trp Asp Met Lys Leu Arg His Leu Gly Leu Gly Lys Thr AspAla Ser 290 295 300 Asp Ser Thr Gln Asn Gln Lys Ser Gly Arg Asn Ser AsnPro Arg Pro 305 310 315 320 Ala Arg Ser 8 2176 DNA Mouse FRZB-1 8aagcctggga ccatggtctg ctgcggcccg ggacggatgc tgctaggatg ggccgggttg 60ctagtcctgg ctgctctctg cctgctccag gtgcccggag ctcaggctgc agcctgtgag 120cctgtccgca tcccgctgtg caagtccctt ccctggaaca tgaccaagat gcccaaccac 180ctgcaccaca gcacccaggc taacgccatc ctggccatgg aacagttcga agggctgctg 240ggcacccact gcagcccgga tcttctcttc ttcctctgtg caatgtacgc acccatttgc 300accatcgact tccagcacga gcccatcaag ccctgcaagt ctgtgtgtga gcgcgcccga 360cagggctgcg agcccattct catcaagtac cgccactcgt ggccggaaag cttggcctgc 420gacgagctgc cggtgtacga ccgcggcgtg tgcatctctc ctgaggccat cgtcaccgcg 480gacggagcgg attttcctat ggattcaagt actggacact gcagaggggc aagcagcgaa 540cgttgcaaat gtaagcctgt cagagctaca cagaagacct atttccggaa caattacaac 600tatgtcatcc gggctaaagt taaagaggta aagatgaaat gtcatgatgt gaccgccgtt 660gtggaagtga aggaaattct aaaggcatca ctggtaaaca ttccaaggga caccgtcaat 720ctttatacca cctctggctg cctctgtcct ccacttactg tcaatgagga atatgtcatc 780atgggctatg aagacgagga acgttccagg ttactcttgg tagaaggctc tatagctgag 840aagtggaagg atcggcttgg taagaaagtc aagcgctggg atatgaaact ccgacacctt 900ggactgggta aaactgatgc tagcgattcc actcagaatc agaagtctgg caggaactct 960aatccccggc cagcacgcag ctaaatcctg aaatgtaaaa ggccacaccc acggactccc 1020ttctaagact ggcgctggtg gactaacaaa ggaaaaccgc acagttgtgc tcgtgaccga 1080ttgtttaccg cagacaccgc gtggctaccg aagttacttc cggtcccctt tctcctgctt 1140cttaatggcg tggggttaga tcctttaata tgttatatat tctgtttcat caatcacgtg 1200gggactgttc ttttgcaacc agaatagtaa attaaatatg ttgatgctaa ggtttctgta 1260ctggactccc tgggtttaat ttggtgttct gtaccctgat tgagaatgca atgtttcatg 1320taaagagaga atcctggtca tatctcaaga actagatatt gctgtaagac agcctctgct 1380gctgcgctta tagtcttgtg tttgtatgcc tttgtccatt tccctcatgc tgtgaaagtt 1440atacatgttt ataaaggtag aacggcattt tgaaatcaga cactgcacaa gcagagtagc 1500ccaacaccag gaagcattta tgaggaaacg ccacacagca tgacttattt tcaagattgg 1560caggcagcaa aataaatagt gttgggagcc aagaaaagaa tattttgcct ggttaagggg 1620cacactggaa tcagtagccc ttgagccatt aacagcagtg ttcttctggc aagtttttga 1680tttgttcata aatgtattca cgagcattag agatgaactt ataactagac atctgttgtt 1740atctctatag ctctgcttcc ttctaaatca aacccattgt tggatgctcc ctctccattc 1800ataaataaat ttggcttgct gtattggcca ggaaaagaaa gtattaaagt atgcatgcat 1860gtgcaccagg gtgttattta acagaggtat gtaactctat aaaagactat aatttacagg 1920acacggaaat gtgcacattt gtttactttt tttcttcctt ttgctttggg cttgtgattt 1980tggtttttgg tgtgtttatg tctgtatttt ggggggtggg taggtttaag ccattgcaca 2040ttcaagttga actagattag agtagactag gctcattggc ctagacatta tgatttgaat 2100ttgtgttgtt taatgctcca tcaagatgtc taataaaagg aatatggttg tcaacagaga 2160cgacaacaac aacaaa 2176 9 325 PRT Human FRZB-1 9 Met Val Cys Gly Ser ProGly Gly Met Leu Leu Leu Arg Ala Gly Leu 1 5 10 15 Leu Ala Leu Ala AlaLeu Cys Leu Leu Arg Val Pro Gly Ala Arg Ala 20 25 30 Ala Ala Cys Glu ProVal Arg Ile Pro Leu Cys Lys Ser Leu Pro Trp 35 40 45 Asn Met Thr Lys MetPro Asn His Leu His His Ser Thr Gln Ala Asn 50 55 60 Ala Ile Leu Ala IleGlu Gln Phe Glu Gly Leu Leu Gly Thr His Cys 65 70 75 80 Ser Pro Asp LeuLeu Phe Phe Leu Cys Ala Met Tyr Ala Pro Ile Cys 85 90 95 Thr Ile Asp PheGln His Glu Pro Ile Lys Pro Cys Lys Ser Val Cys 100 105 110 Glu Arg AlaArg Gln Gly Cys Glu Pro Ile Leu Ile Lys Tyr Arg His 115 120 125 Ser TrpPro Glu Asn Leu Ala Cys Glu Glu Leu Pro Val Tyr Asp Arg 130 135 140 GlyVal Cys Ile Ser Pro Glu Ala Ile Val Thr Ala Asp Gly Ala Asp 145 150 155160 Phe Pro Met Asp Ser Ser Asn Gly Asn Cys Arg Gly Ala Ser Ser Glu 165170 175 Arg Cys Lys Cys Lys Pro Ile Arg Ala Thr Gln Lys Thr Tyr Phe Arg180 185 190 Asn Asn Tyr Asn Tyr Val Ile Arg Ala Lys Val Lys Glu Ile LysThr 195 200 205 Lys Cys His Asp Val Thr Ala Val Val Glu Val Lys Glu IleLeu Lys 210 215 220 Ser Ser Leu Val Asn Ile Pro Arg Asp Thr Val Asn LeuTyr Thr Ser 225 230 235 240 Ser Gly Cys Leu Cys Pro Pro Leu Asn Val AsnGlu Glu Tyr Ile Ile 245 250 255 Met Gly Tyr Glu Asp Glu Glu Arg Ser ArgLeu Leu Leu Val Glu Gly 260 265 270 Ser Ile Ala Glu Lys Trp Lys Asp ArgLeu Gly Lys Lys Val Lys Arg 275 280 285 Trp Asp Met Lys Leu Arg His LeuGly Leu Ser Lys Ser Asp Ser Ser 290 295 300 Asn Ser Asp Ser Thr Gln SerGln Lys Ser Gly Arg Asn Ser Asn Pro 305 310 315 320 Arg Gln Ala Arg Asn325 10 1893 DNA Human FRZB-1 10 ggcggagcgg gccttttggc gtccactgcgcggctgcacc ctgccccatc tgccgggatc 60 atggtctgcg gcagcccggg agggatgctgctgctgcggg ccgggctgct tgccctggct 120 gctctctgcc tgctccgggt gcccggggctcgggctgcag cctgtgagcc cgtccgcatc 180 cccctgtgca agtccctgcc ctggaacatgactaagatgc ccaaccacct gcaccacagc 240 actcaggcca acgccatcct ggccatcgagcagttcgaag gtctgctggg cacccactgc 300 agccccgatc tgctcttctt cctctgtgccatgtacgcgc ccatctgcac cattgacttc 360 cagcacgagc ccatcaagcc ctgtaagtctgtgtgcgagc gggcccggca gggctgtgag 420 cccatactca tcaagtaccg ccactcgtggccggagaacc tggcctgcga ggagctgcca 480 gtgtacgaca ggggcgtgtg catctctcccgaggccatcg ttactgcgga cggagctgat 540 tttcctatgg attctagtaa cggaaactgtagaggggcaa gcagtgaacg ctgtaaatgt 600 aagcctatta gagctacaca gaagacctatttccggaaca attacaacta tgtcattcgg 660 gctaaagtta aagagataaa gactaagtgccatgatgtga ctgcagtagt ggaggtgaag 720 gagattctaa agtcctctct ggtaaacattccacgggaca ctgtcaacct ctataccagc 780 tctggctgcc tctgccctcc acttaatgttaatgaggaat atatcatcat gggctatgaa 840 gatgaggaac gttccagatt actcttggtggaaggctcta tagctgagaa gtggaaggat 900 cgactcggta aaaaagttaa gcgctgggatatgaagcttc gtcatcttgg actcagtaaa 960 agtgattcta gcaatagtga ttccactcagagtcagaagt ctggcaggaa ctcgaacccc 1020 cggcaagcac gcaactaaat cccgaaatacaaaaagtaac acagtggact tcctattaag 1080 acttacttgc attgctggac tagcaaaggaaaattgcact attgcacatc atattctatt 1140 gtttactata aaaatcatgt gataactgattattacttct gtttctcttt tggtttctgc 1200 ttctctcttc tctcaacccc tttgtaatggtttgggggca gactcttaag tatattgtga 1260 gttttctatt tcactaatca tgagaaaaactgttcttttg caataataat aaattaaaca 1320 tgctgttacc agagcctctt tgctgagtctccagatgtta atttactttc tgcaccccaa 1380 ttgggaatgc aatattggat gaaaagagaggtttctggta ttcacagaaa gctagatatg 1440 ccttaaaaca tactctgccg atctaattacagccttattt ttgtatgcct tttgggcatt 1500 ctcctcatgc ttagaaagtt ccaaatgtttataaaggtaa aatggcagtt tgaagtcaaa 1560 tgtcacatag gcaaagcaat caagcaccaggaagtgttta tgaggaaaca acacccaaga 1620 tgaattattt ttgagactgt caggaagtaaaataaatagg agcttaagaa agaacatttt 1680 gcctgattga gaagcacaac tgaaaccagtagccgctggg gtgttaatgg tagcattctt 1740 cttttggcaa tacatttgat ttgttcatgaatatattaat cagcattaga gaaatgaatt 1800 ataactagac atctgctgtt atcaccatagttttgtttaa tttgcttcct tttaaataaa 1860 cccattggtg aaagtcaaaa aaaaaaaaaaaaa 1893

It is claimed:
 1. A substantially pure protein characterized by aphysiologically active form and comprising an amino acid sequenceencoded by the DNA of SEQ ID NO:
 2. 2. The protein as in claim 1 havingneurotrophic, growth or differentiation factor activity.
 3. Acomposition comprising the protein of claim 1 and a physiologicallyacceptable carrier with which the peptide is admixed.
 4. Anoligonucleotide construct comprising a sequence coding for a protein andan expression vector operatively linked therewith, the protein havingneurotrophic, growth or differentiation factor activity and beingexpressible from SEQ ID NO:
 2. 5. The construct as in claim 4 whereinthe expression vector is a mammalian or viral expression vector.
 6. Asubstantially pure protein characterized by a physiologically activeform and comprising an amino acid sequence encoded by the DNA of SEQ IDNO: 4 1 SEQ ID NO: 8, or SEQ ID NO:
 10. 7. The protein as in claim 6having neurotrophic, growth or differentiation factor activity.
 8. Acomposition comprising the protein of claim 6 and a physiologicallyacceptable carrier with which the protein is admixed.
 9. Anoligonucleotide construct comprising a sequence coding for a protein andan expression vector operatively linked therewith, the protein beingexpressible from SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO:10.
 10. Theconstruct as in claim 9 wherein the protein is expressible in solubleform.
 11. The construct as in claim 9 wherein the expression vector is amammalian or viral expression vector.
 12. A complex comprising asubstantially pure frzb-1 protein complexed with at least one Wntprotein.
 13. A substantially pure protein characterized by aphysiologically active form and comprising an amino acid sequenceencoded by the DNA of SEQ ID NO:
 6. 14. The protein as in claim 13having mesoderm differentiation activity.
 15. A composition comprisingthe protein of claim 13 and a physiologically acceptable carrier withwhich the protein is admixed.